Complex mixtures such as biological samples can contain up to 30,000 different proteins which need to be separated and identified for further analysis. In proteome analysis, high resolution separation of complex protein mixtures requires the development of novel techniques which minimize separation times, are easy to use, result in a high degree of purity and allow for further analysis of the compound(s) of interest extracted from the sample without unnecessary additional purification steps.
2D-gel electrophoresis is one technique which is capable of separating such complex biological samples (Wilkins, M. R. et al., Proteome Research: New Frontiers in Functional Genomics; Springer, 1997). With 2D-gel electrophoresis, proteins are separated first by an isoelectric focusing (IEF) step according to their isoelectric point. Secondly, proteins are separated as a function of their molecular mass by a polyacrylamide gel electrophoresis (PAGE) step. The result is a two-dimensional image in which each visible spot corresponds to a specific protein. If further analysis of a protein is required, for example, analysis of peptide composition or biological activity, then the protein has to be first extracted from the gel matrix before it can be analysed with the appropriate method to obtain the desired information.
Methods have been developed to extract proteins from a polyacrylamide 2D-gel, one such method consisting of cutting the gel around the protein spot and extracting it in a wet chemical step. With this technique there is a high probability that the protein will be denatured, modified or even lost during its retrieval. Another technique is electroblotting, which is very time-consuming.
Once a protein is extracted from the gel, the most powerful analytical technique is mass spectrometry. Using this technique, it is not only possible to analyse the peptide composition of proteins, but also to compare the obtained peptide map to other protein data compiled in data banks by several bioinformatical institutions. In mass spectroscopy (MS), the purity of a sample is critical. If a sample contains impurities such as salt, this is not directly amenable to MS analysis. Such a sample would require desalting before MS analysis by means such as a dialysis procedure. Another way of avoiding undesired compounds in the sample is to use a direct laser desorption technique from the 2D-gel (Ogorzalek Loo R. R., et al., Analytical Chemistry, 1996, 68, 1910-1917) or an electroblotted 2D-gel (Eckerskom, C. et.al., Analytical Chemistry, 1997, 69, 2888-2892; Strupat, K. et al. Analytical Chemistry, 1994, 66, 464-470). All of these additional purification steps complicate the analysis procedure and are time-consuming.
While separation of compounds in complex mixtures is possible with 2D-gel electrophoresis, there persists the problem of the numerous impurities which remain together with the compounds desired for analysis, removal of which is laborious. The major problem with 2D gel-electrophoresis is that the compound of interest is trapped within a gel and must be extracted and further purified before it can be analysed.
Another method of separating complex biological samples is by isoelectric separation for example by iso-electric focusing (IEF) (Righetti, P. G., J. Biochem Biophys Methods, 1988 16:99-108). IEF is a technique of electrophoresis whereby compounds can be separated on the basis of charge within a pH gradient. In general, there are two major types of iso-electric focusing systems: (i) free flowing buffered systems and (ii) immobilised buffered systems.
(i) Free Flowing Buffering Systems
All free flowing systems are based on the use of a buffer, usually carrier ampholytes or isoelectric buffers such as amino acids. For example, a continuous free flow device has been demonstrated by Soulet, N. et al. (Electrophoresis, 1998, 19, 1294-1299). In this device, a pH gradient is created in a flat chamber using carrier ampholytes and a potential gradient perpendicular to the carrier flow direction. The sample is continuously injected and partitioned at the end of the device in discrete fractions. Although the pH gradient was stable over several hours, a complete separation of bovine serum albumin and alpha-lactalbumin could not be achieved. Some major drawbacks of this system are that it is not able to separate compounds which have close p1 values, that it takes several hours for the separation to occur and that it also uses carrier ampholytes which need to be removed before further analysis of the desired compounds is possible.
In isoelectric split-flow thin (SPLITT) fractionation, no pH gradient is established, but the separation principle is based on the charge that proteins exhibit depending on their isoelectric point (pI) in buffers of different pH. A potential is applied to a flow cell using adequate outlet and/or inlet splitters to separate the protein fractions. Two component protein mixtures have been successfully separated (Fuh, C. B. and Giddings, J. C., Separation Science and Technology, 1997, 32, 2945-2967), but this system exhibits some drawbacks when complex protein samples have to be analysed and when the isoelectric points (PI) of proteins are very close (pI differences less than 0.1 pH unit are not possible to separate using this method).
Many recycling isoelectric systems are based on the physical separation of compartments with different pH by means of membranes or screens. Some of them have been reviewed in the literature (Bier, M. Electrophoresis, 1998,19,1057-1063; Krivankova, L. et al., Electrophoresis, 1998, 19, 1064-1074). One of the most common preparative approaches to recycling free-flow electrophoresis is the Rotofor apparatus, commercialised by BioRad. In a tube-like apparatus where compartments are defined by a screening material, the pH gradient is established using special ampholytes, the so-called Rotolytes. Gravity problems in free flow electrophoresis are overcome by the rotation of the separation compartments. This device has been successfully applied to the preparative scale. A modification of this approach is the tangential electrophoretic apparatus from Bier, (U.S. Pat. No. 5,540,826). Here, the different compartments are arranged in such a manner that an array of multi-channels is separated from a second array of multi-channels slightly displaced through a single screen. An electrical field is applied perpendicularly to the channels which enables an electrophoretic serpentine pathway through the channels. The pH in the channels is fixed by ampholytes and recycling is possible with independent inlet and outlet ports at every channel. The major disadvantages of this system are that the device has a complicated construction of multi-channels through which the solution must flow and that the compound(s) of interest remain(s) in an ampholyte solution which needs to be removed before further analysis of the desired compound or compounds is possible.
In most solution-buffered systems, the analyte is mixed with a running buffer and several strategies of fluid handling are presented to either fractionate or desalt the sample or to work in a non convective and/or low water diffusion medium. All these isoelectric focusing devices have a major disadvantage in terms of further analysis of compounds. They all contain in the final separated fraction a certain amount of undesired buffering species or ampholytes.
(ii) Immobilized Buffering System
In most immobilized buffering systems, there is a major disadvantage in terms of further analysis of compounds since the final separated fraction is trapped in a gel or membrane.
There is a device developed by Righetti and Faupel (Righetti, P. G. et al. Journal of Chromatography, 1989, 475, 293-309) which is based on a technique known as “segmented immobilized pH gradients”. The device is composed of multiple compartments sandwiched between an anodic and a cathodic reservoir separated by immobiline isoelectric membranes, allowing the recovery of proteins in an ampholyte-free solution. This device can be composed of several compartments separated by immobiline gels stabilised by membranes. The separation of fractions is achieved in such a way that the protein stops migrating in an electrical field in between two immobiline membranes, wherein one membrane establishes a pH higher than the protein's pI and the other a pH lower than it. There are several disadvantages of this apparatus: the use of multiple compartments, multiple immobilized membranes and segmented pH gradients.